The goal of project 2 is to understand the role of WW domain proteins in the regulation of renal Na + absorption via the epithelial Na + channel, ENaC. ENaC forms the pathway for Na + absorption in the kidney collecting duct and other epithelia. Thus, this channel plays a critical role in Na + homeostasis and blood pressure control. Mechanisms that control the expression of ENaC at the cell surface play an important role in the regulation of epithelial Na + absorption. A key sequence is the PY motif, located in the C-termini of ENaC subunits. Mutation or deletion of this sequence increases the number of ENaC channels at the cell surface, causing an inherited form of hypertension (Liddle's syndrome). PY motifs mediate protein interactions by binding to WW domains, implicating a critical role for WW domain proteins. In previous work, we and others found that a WW domain protein (Nedd4) decreased Na + current by binding to ENaC and targeting the channel for degradation. However, the role of Nedd4 in the kidney collecting duct is unknown. Moreover, it is clear that Nedd4 is part of a large family of Nedd4-related proteins. Thus, we will use a systematic approach to test the hypothesis that WW domain proteins modulate ENaC-mediated Na + absorption in the collecting duct. In Specific Aim 1, we will determine which WW domain proteins are candidates, based on their expression patterns, their binding to ENaC subunits, and their ability to inhibit ENaC in heterologous expression systems. In Specific Aim 2, we will extend these results to the kidney collecting duct, and other epithelia that express ENaC. We will test the hypothesis that specific WW domain proteins interact with ENaC, and inhibit Na + current in M1 and H441 cells, derived from the kidney collecting duct and lung, respectively. In Specific Aim 3, we will identify the sequences in the WW domain proteins and ENaC that mediate the interaction between these proteins, and that are required for down-stream events leading to channel degradation. First, we will test which WW domains and PY motifs are required. Second, we will test the role of WW domain proteins, and specific sequences, in the Ca2+-dependent regulation of ENaC. Third, we will test the hypothesis that polymorphisms in ENaC and WW domain proteins alter binding and/or channel inhibition. Because these ENaC polymorphisms were identified in hypertensive populations, this work could have important implications for our understanding of hypertension, and other disorders of Na + homeostasis.